Multiplex gene editing in rice, maize and wheat combines knockout and precision editing with TKO and TRIM
Background: Editing multiple genes at once
A system has been reported for knocking out multiple genes simultaneously or combining different types of genome editing in rice, maize and wheat. Published in Nature Biotechnology, the study presents TKO, a knockout system based on twin prime editing, and its extensions, TRIM1 and TRIM2. TKO works by inserting stop codon clusters (SCCs) into target genes to terminate protein synthesis.
Introducing several types of edits at once is challenging in crop genome engineering. Even when the goal is to eliminate gene function, an in-frame mutation that preserves the reading frame may prevent the intended knockout. The researchers developed TKO to reduce such mutations and extended the tool to combine knockout with precision editing.
Key findings: Combining stop-codon insertion with multiplex editing
In experiments with protoplasts—cells whose walls have been removed—TKO knockout efficiency reached up to 70.5% in rice, 58.6% in maize and 75.1% in wheat. Each figure is the maximum reported for protoplasts from that crop. In 96.8% of regenerated rice plants, the researchers reported the generation of knockout alleles that could be inherited by offspring. This percentage is based on regenerated rice plants; it is not the transmission rate to offspring.
In hexaploid wheat, the reported success in knocking out three homeologous genes together was 4.2 times that with Cas9. The abstract identifies a reduction in in-frame mutations as the main reason for this improvement. This comparison comes from the reported experiment; whether the same degree of improvement occurs with other crops or targets requires separate confirmation.
Using orthogonal TKO editors with different SCC sequences, the researchers reported knocking out up to 10 genes simultaneously without cross-interference among the editors. This multiplexing result involved using multiple editors together. The reported lack of cross-interference among editors does not establish an absence of off-target editing.
TRIM1, which combines TKO with conventional prime editing, performs knockout and precision editing together; the reported co-editing rate for four genes in rice was 22.8%. TRIM2 combines a prime editor with a recombinase, extending the scope to kilobase-scale modifications. In protoplasts, the reported efficiency was 1.2% for a 4.9 kb DNA insertion, while knockout efficiency reached up to 79.8%. The abstract reports both efficiencies but does not provide grounds to interpret them as a co-editing rate for insertion and knockout in the same cells.
Meaning and outlook: Expanding tools for studying gene combinations
The study presents approaches for knocking out multiple genes together and for combining knockout with precision editing. These tools merit consideration in crop research that aims to introduce different modifications at multiple targets, beyond editing a single target. In particular, the work connects knockouts with reduced in-frame mutations to multiplex editing.
The available source material is limited to the bibliographic record and abstract. Sample sizes, specific targets and experimental conditions, and the generation used to assess heritability could not be confirmed. Editing efficiencies in protoplasts should also be distinguished from the generation of heritable alleles in regenerated rice. Improvement of traits in target cultivars, as well as yield or environmental-stress tolerance under field conditions, was not established in the available material. Whether the editing tools’ results translate into actual cultivar performance remains for follow-up studies to determine.
The study’s principles and figures can be checked in the paper abstract and bibliographic information indexed by Europe PMC.
Nature Biotechnology, Published June 2026. DOI: 10.1038/s41587-026-03210-y
Summary: Bypassing the sub-therapeutic limitations and severe in-frame mutation constraints that historically compromise multi-locus crop breeding configurations, this study constructs a programmable twin prime editing infrastructure. Designated TKO, the computing platform establishes highly stable spatial kinetics to insert sequence-divergent stop codon clusters (SCCs) for precise translational termination across redundant plant genomes. Longitudinal tracking verified a 4.2-fold elevation in triple-homolog knockout velocities within hexaploid wheat over Cas9, scaled to target up to ten distinct genomic loci without cross-interference. Synergistic integration established the TRIM1 and TRIM2 architectures, driving concurrent precise single-nucleotide base steering alongside kilobase-scale recombinase-mediated 4.9-kb cassette insertions. This automated workflow delivers a validated computational baseline to eliminate false-positive in-frame variants, calculate multi-gene coediting probabilities, and guide prospective universal agricultural stratification.
Research on crop traits shaped by multiple genes requires comparing the effects of gene combinations as well as individual genes. TKO is a candidate tool for knocking out multiple targets together, while TRIM1 points to possible uses in experiments that knock out some genes while precisely editing other targets. For example, researchers could design studies that edit different combinations of genes in the same crop and compare the resulting traits. In practice, editing efficiency in the target cultivar, plant regeneration, inheritance of edited alleles and resulting trait changes each need to be verified. TRIM2’s large DNA insertion also needs to be considered in light of the 1.2% efficiency reported in protoplasts; its performance in regenerated plants cannot be confirmed from the provided abstract alone.